利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学
Summary
蛋白质-蛋白质相互作用对生物系统至关重要, 对结合动力学的研究为蛋白质复合物的动力学和功能提供了见解。我们描述了一种利用荧光共振能量转移和停止流动技术量化蛋白质复合物动力学参数的方法。
Abstract
蛋白质是生物系统的主要操作人员, 它们通常与其他大分子或小分子相互作用, 以发挥其生物功能。这种相互作用可以是高度动态的, 这意味着相互作用的子单位以一定的速度不断地联系和分离。虽然使用定量下拉式等技术测量结合亲和力可以揭示相互作用的强度, 但研究结合动力学可以提供有关相互作用发生的速度以及每个复合物存在多长时间的见解。此外, 在存在蛋白质交换因子或药物等附加因素的情况下测量相互作用的动力学, 有助于揭示相互作用受另一个因素调节的机制, 为生物和医学研究的进步。在这里, 我们描述了一个协议, 用于测量具有高内在关联率并可由另一种蛋白质快速分离的蛋白质复合体的结合动力学。该方法利用荧光共振能量转移报告蛋白质复合物的体外形成, 并能实时监测停止流动的荧光仪上的蛋白质复合物的快速关联和离解。利用该方法, 对蛋白质复合物的关联和离解速率常数进行了定量。
Introduction
生物活动最终是由蛋白质进行的, 其中大部分蛋白质与其他蛋白质相互作用, 以获得适当的生物功能。使用计算方法, 人类蛋白质相互作用的总量估计为约 650, 000,1, 而这些相互作用的中断往往导致疾病2。由于其在控制细胞和组织过程中的重要作用, 研究蛋白质-蛋白质相互作用的方法已被开发出来, 如酵母-双杂交, 双分子荧光互补, 分裂荧光酶互补, 联合免疫沉淀检测3。虽然这些方法善于发现和确认蛋白质-蛋白质相互作用, 但它们通常是非定量的, 因此提供的相互作用的蛋白质伙伴之间的亲和力有限。定量拉下可以用来测量结合亲和力 (例如, 离解常数kd), 但它不测量结合的动力学, 也不能在 k d 由于不足而非常低的情况下应用信噪比4。表面等离子体共振 (spr) 光谱量化了结合动力学, 但它需要一个特定的表面和在表面的一个反应物的固定, 这可能会改变反应物5的结合性能。此外, spr 很难测量快速关联和离解率5, 使用 spr 来表征蛋白质复合体中交换蛋白质亚基的事件是不合适的。在这里, 我们描述了一种方法, 允许测量在毫秒时间尺度上的蛋白质复杂组装和拆卸速率。该方法对于确定 c ullin-a ssociated-edd8-d 同体蛋白 1 (cand1) 作为 f- 盒蛋白交换因子6,7的作用至关重要.
cand1 调节 Skp1•Cul1•F 盒蛋白 (scf) e3 脂类的动态, e3 配体属于库林-岭泛素脂质的大家族。scfs 由结合 ing 域蛋白 rbx1 的 cullin cullin 和一种可互换的 f-箱蛋白组成, 后者通过适配器蛋白 skp18 招募基质并将 cullin 结合。作为 e3 连接酶, scf 催化泛素与底物的结合, 当底物被 f-盒蛋白吸收, 当 cul1 被泛素样蛋白 nedd89 修饰时, 它就会被激活。cand1 结合未经修饰的 cul1, 在结合时, 它破坏了 Skp1•F 盒蛋白与 cul1 的联系, 以及 ned8 与 cul1、11、12、13的结合。因此, cand1 似乎是体外 scf 活性的抑制剂, 但体内的 cand1 缺乏引起了缺陷, 表明 cand1 在调节体内 scf 活性方面发挥了积极作用 14、15、16,17. 这一悖论最终通过一项定量研究得到了解释, 该研究揭示了 cul1、cand1 和 Skp1•F 盒蛋白之间的动态相互作用。利用荧光共振能量传递 (fret) 检测 scf 和 Cul1•Cand1 配合物的形成, 分别检测出关联和离解速率常数 (分别为 kon 和k)。单独测量。测量结果表明, cand1 和 Skp1•F 盒蛋白与 cul1 的复合体极其紧密, 但 scf 的k含量因 cand1 而急剧增加, Cul1•Cand1 的k含量大幅增加由 Skp1•F 盒蛋白6,7。这些结果为定义 cand1 作为蛋白质交换因子的作用提供了初步和关键的支持, 该因子通过从旧的 scf 复合物中回收 cul1 来催化新的 scf 复合物的形成。
本文介绍了利用 fret 法研究 Cul1•Cand1 复合物7动力学的方法, 并将同样的原理应用于各种生物分子的动力学研究。当供体被适当的波长激发, 而具有激发谱重叠供体发射光谱的受体存在于10-100 的距离内时, 就会发生 fret。激发态转移到受体, 从而降低供体强度, 增加受体强度18。fret (e) 的效率取决于 förster 半径 (r0) 和供体与受体荧光 (r) 之间的距离, 定义为: e = r0 6/(r0 )6 + r6)。förster 半径 (r0) 取决于几个因素, 包括偶极子角方向、供体受体对的光谱重叠和使用的解 19.为了将 fret 检测应用于停止流式荧光仪上, 该方法实时监测供体排放的变化, 并能够测量快速k开和关,有必要建立高效的 fret, 以实现从而显著减少了捐助方的排放。因此, 通过选择合适的对荧光染料和在目标蛋白上的位点来连接染料来设计高效的 fret 是非常重要的, 将在本方案中进行讨论。
Protocol
Representative Results
Discussion
fret 是一种物理现象, 对研究和理解生物系统非常感兴趣。在这里, 我们提出了一个测试和使用 fret 研究两种相互作用的蛋白质的结合动力学的方案。在设计 fret 时, 我们考虑了三个主要因素: 供体发射和受体激励之间的光谱重叠、两个荧光体之间的距离以及荧光的偶极子方向 28.为了选择 fret 的荧光体, 我们对荧光的激发和发射光谱进行了叠加, 并寻找了其发射峰?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢加州理工学院 (shu-ou shan) 就 fret 检测的发展进行了富有见地的讨论。硕士、y. z. 和 x. l. 由普渡大学向 y. z. 提供的创业资金资助 X.L.This 工作部分由普渡大学植物生物学中心的种子赠款提供。
Materials
| Anion exchange chromatography column | GE Healthcare | 17505301 | HiTrap Q FF anion exchange chromatography column |
| Benchtop refrigerated centrifuge | Eppendorf | 2231000511 | |
| BL21 (DE3) Competent Cells | ThermoFisher Scientific | C600003 | |
| Calcium Chloride | Fisher Scientific | C78-500 | |
| Cation exchange chromatography column | GE Healthcare | 17505401 | HiTrap SP Sepharose FF |
| Desalting Column | GE Healthcare | 17085101 | |
| Floor model centrifuge (high speed) | Beckman Coulter | J2-MC | |
| Floor model centrifuge (low speed) | Beckman Coulter | J6-MI | |
| Fluorescence SpectraViewer | ThermoFisher Scientific | https://www.thermofisher.com/us/en/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html | |
| FluoroMax fluorimeter | HORIBA | FluoroMax-3 | |
| FPLC | GE Healthcare | 29018224 | |
| GGGGAMC peptide | New England Peptide | custom synthesis | |
| Glutathione beads | GE Healthcare | 17075605 | |
| Glycerol | Fisher Scientific | G33-500 | |
| HEPES | Fisher Scientific | BP310-100 | |
| Isopropyl-β-D-thiogalactoside (IPTG) | Fisher Scientific | 15-529-019 | |
| LB Broth | Fisher Scientific | BP1426-500 | |
| Ni-NTA agarose | Qiagen | 30210 | |
| Ovalbumin | MilliporeSigma | A2512 | |
| pGEX-4T-2 vector | GE Healthcare | 28954550 | |
| Protease inhibitor cocktail | MilliporeSigma | 4693132001 | |
| Reduced glutathione | Fisher Scientific | BP25211 | |
| Refrigerated shaker | Eppendorf | M1282-0004 | |
| Rosetta Competent Cells | MilliporeSigma | 70953-3 | |
| Size exclusion chromatography column | GE Healthcare | 28990944 | Superdex 200 10/300 GL column |
| Sodium Chloride (NaCl) | Fisher Scientific | S271-500 | |
| Stopped-flow fluorimeter | Hi-Tech Scientific | SF-61 DX2 | |
| TCEP·HCl | Fisher Scientific | PI20490 | |
| Thrombin | MilliporeSigma | T4648 | |
| Tris Base | Fisher Scientific | BP152-500 | |
| Ultrafiltration membrane | MilliporeSigma | UFC903008 | Amicon Ultra-15 Centrifugal Filter Units, Ultra-15, 30,000 NMWL |
References
- Stumpf, M. P. H., et al. Estimating the size of the human interactome. Proceedings of the National Academy of Sciences of the United States of America. 105 (19), 6959-6964 (2008).
- Kuzmanov, U., Emili, A. Protein-protein interaction networks: probing disease mechanisms using model systems. Genome Medicine. 5 (4), 37 (2013).
- Titeca, K., Lemmens, I., Tavernier, J., Eyckerman, S. Discovering cellular protein-protein interactions: Technological strategies and opportunities. Mass Spectrometry Reviews. , 1-33 (2018).
- Lapetina, S., Gil-Henn, H. A guide to simple, direct, and quantitative in vitro binding assays. Journal of Biological Methods. 4 (1), 62 (2017).
- Zheng, X., Bi, C., Li, Z., Podariu, M., Hage, D. S. Analytical methods for kinetic studies of biological interactions: A review. Journal of Pharmaceutical and Biomedical Analysis. 113, 163-180 (2015).
- Pierce, N. W., et al. Cand1 promotes assembly of new SCF complexes through dynamic exchange of F box proteins. Cell. 153 (1), 206-215 (2013).
- Liu, X., Reitsma, J. M., Mamrosh, J. L., Zhang, Y., Straube, R., Deshaies, R. J. Cand1-Mediated Adaptive Exchange Mechanism Enables Variation in F-Box Protein Expression. Molecular Cell. 69 (5), 773-786 (2018).
- Zheng, N., et al. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex. Nature. 416 (6882), 703-709 (2002).
- Kleiger, G., Deshaies, R. Tag Team Ubiquitin Ligases. Cell. 166 (5), 1080-1081 (2016).
- Zheng, J., et al. CAND1 binds to unneddylated CUL1 and regulates the formation of SCF ubiquitin E3 ligase complex. Molecular Cell. 10 (6), 1519-1526 (2002).
- Hwang, J. W., Min, K. W., Tamura, T. A., Yoon, J. B. TIP120A associates with unneddylated cullin 1 and regulates its neddylation. FEBS Letters. 541 (1-3), 102-108 (2003).
- Min, K. W., Hwang, J. W., Lee, J. S., Park, Y., Tamura, T., Yoon, J. B. TIP120A associates with cullins and modulates ubiquitin ligase activity. Journal of Biological Chemistry. 278 (18), 15905-15910 (2003).
- Goldenberg, S. J., et al. Structure of the Cand1-Cul1-Roc1 complex reveals regulatory mechanisms for the assembly of the multisubunit cullin-dependent ubiquitin ligases. Cell. 119 (4), 517-528 (2004).
- Chuang, H. W., Zhang, W., Gray, W. M. Arabidopsis ETA2, an apparent ortholog of the human cullin-interacting protein CAND1, is required for auxin responses mediated by the SCF(TIR1) ubiquitin ligase. Plant Cell. 16 (7), 1883-1897 (2004).
- Feng, S., et al. Arabidopsis CAND1, an Unmodified CUL1-Interacting Protein, Is Involved in Multiple Developmental Pathways Controlled by Ubiquitin/Proteasome-Mediated Protein Degradation. the Plant Cell Online. 16 (7), 1870-1882 (2004).
- Cheng, Y., Dai, X., Zhao, Y. AtCAND1, a HEAT-repeat protein that participates in auxin signaling in Arabidopsis. Plant Physiology. 135 (June), 1020-1026 (2004).
- Lo, S. -. C., Hannink, M. CAND1-Mediated Substrate Adaptor Recycling Is Required for Efficient Repression of Nrf2 by Keap1. Molecular and Cellular Biology. 26 (4), 1235-1244 (2006).
- Okamoto, K., Sako, Y. Recent advances in FRET for the study of protein interactions and dynamics. Current Opinion in Structural Biology. 46, 16-23 (2017).
- Hussain, S. A. An introduction to fluorescence resonance energy transfer (FRET). arXiv preprint. , (2009).
- Li, T., Pavletich, N. P., Schulman, B. A., Zheng, N. High-level expression and purification of recombinant SCF ubiquitin ligases. Methods in Enzymology. 398 (1996), 125-142 (2005).
- Popp, M. W., Antos, J. M., Grotenbreg, G. M., Spooner, E., Ploegh, H. L. Sortagging: A versatile method for protein labeling. Nature Chemical Biology. 3 (11), 707-708 (2007).
- Antos, J. M., Ingram, J., Fang, T., Pishesha, N., Truttmann, M. C., Ploegh, H. L. Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation. Current Protocols in Protein Science. 89, (2017).
- Froger, A., Hall, J. E. Transformation of Plasmid DNA into E. coli Using the Heat Shock Method. Journal of Visualized Experiments. , (2007).
- Simpson, R. J. SDS-PAGE of Proteins. Cold Spring Harbor Protocols. 2006 (1), (2006).
- Kleiger, G., Saha, A., Lewis, S., Kuhlman, B., Deshaies, R. J. Rapid E2-E3 Assembly and Disassembly Enable Processive Ubiquitylation of Cullin-RING Ubiquitin Ligase Substrates. Cell. 139 (5), 957-968 (2009).
- Biro, F. N., Zhai, J., Doucette, C. W., Hingorani, M. M. Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein. Journal of Visualized Experiments. (37), 2-8 (2010).
- Patel, J. T., Belsham, H. R., Rathbone, A. J., Friel, C. T. Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins. Journal of Visualized Experiments. (92), 1-6 (2014).
- Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z., Chu, J. A guide to fluorescent protein FRET pairs. Sensors (Switzerland). 16 (9), 1-24 (2016).
- Chen, A. K., Cheng, Z., Behlke, M. A., Tsourkas, A. Assessing the sensitivity of commercially available fluorophores to the intracellular environment. Analytical Chemistry. 80 (19), 7437-7444 (2008).
- Lin, C. T., Rorabacher, D. B. Mathematical approach for stopped-flow kinetics of fast second-order reactions involving inhomogeneity in the reaction cell. Journal of Physical Chemistry. 78 (3), 305-308 (1974).
- Toseland, C. P., Geeves, M. A. Rapid Reaction Kinetic Techniques. Fluorescent Methods for Molecular Motors. , 49-65 (2014).
- Adams, S. R., et al. New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications. Journal of the American Chemical Society. 124 (21), 6063-6076 (2002).
- Lin, C. W., Ting, A. Y. Transglutaminase-catalyzed site-specific conjugation of small-molecule probes to proteins in vitro and on the surface of living cells. Journal of the American Chemical Society. 128 (14), 4542-4543 (2006).
- Yin, J., et al. Genetically encoded short peptide tag for versatile protein labeling by Sfp phosphopantetheinyl transferase. Proceedings of the National Academy of Sciences of the United States of America. 102 (44), 15815-15820 (2005).
- Diaspro, A., Chirico, G., Usai, C., Ramoino, P., Dobrucki, J. Photobleaching. Handbook Of Biological Confocal Microscopy. , 690-702 (2006).
- Aoki, K., Kamioka, Y., Matsuda, M. Fluorescence resonance energy transfer imaging of cell signaling from in vitro to in vivo: Basis of biosensor construction, live imaging, and image processing. Development, Growth & Differentiation. 55 (4), 515-522 (2013).
- Kilic, S., et al. Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α. Nature Communications. 9 (1), (2018).
- Shen, K., Arslan, S., Akopian, D., Ha, T., Shan, S. O. Activated GTPase movement on an RNA scaffold drives co-translational protein targeting. Nature. 492 (7428), 271-275 (2012).
- Bajar, B. T., et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Scientific Reports. 6 (February), 1-12 (2016).
